Thursday, December 24, 2009

Working on my Master’s degree has made me yen for more letters after my name, so I’ve been doing some spare-time reading on subjects that might yield PhD-type projects. My putative interest is in development of the stress system in young dogs. The idea is that if a dog’s stress system develops poorly, whether through bad genetics or a bad early environment, then that dog is more likely to bite people when it grows up. The more we know about how their stress system develops, the more we can know about how to grow healthy dogs with good bite inhibition.

For several months I thrashed around in the literature, reading about development of the stress system in rodents (about whom we know quite a bit, because we are more willing to do experiments on them than on dogs), and reading about socialization periods in dogs. It was hard to find good direction, and I wasn’t quite sure where to start. Recently I have had a breakthrough, however.

First, some orientation. You are walking through the woods. You see a shape on the ground. Your brain interprets the shape: long, thin. Your amygdala (part of the limbic system of your brain) yells SCARY SHAPE SCARY SHAPE and you get a blast of adrenaline in your system. Half a second later your cortex (the thinking, conscious part of your brain) catches up: hey, that looks like a snake. Your hypothalamus (which deals with a lot of hormone regulation) sends a message to your pituitary (which releases a lot of your hormones), and the pituitary releases a hormone which travels down to your adrenals, near your kidneys. Your adrenals release our old friend cortisol, which gets into your blood and tells your body that you are having a stressful experience. Cortisol, you of course remember, is what I like to extract from the saliva of dogs to tell if they are unhappy about being stuck in a noisy hospital run. This whole system is what I’ve been referring to as the “stress system,” more properly called the HPA (hypothalamic-pituitary-adrenal) axis.

If you were a rat or mouse, instead of releasing cortisol, your adrenals would release corticosterone. It is a very similar hormone with similar effects. Dogs actually release equal parts cortisol and corticosterone, but we just study their cortisol levels. I still haven’t figured out why we chose cortisol to focus on in them; there are a lot of tools available for studying cortisol, since humans make it primarily, but also a lot for studying corticosterone, since we study rodents quite a bit.

Now, to get back to my recent reading, very young animals don’t get as frightened by scary things as slightly more mature animals or adults. This phenomenon has been studied intensively in the rat: rats younger than two weeks of age don’t show this corticosterone spike when exposed to something upsetting. This is called the “stress hyporesponsive period,” or SHRP.[1] There has been work on what part of the HPA system is responsible for this blunted response: the amygdala? The hypothalamus? The pituitary? Or are the adrenals themselves not responsive yet?

A good way to stress out an infant rat is to expose it to the odor of an adult male rat. Left to their own devices, adult males will happily eat infants, so the young rats are quite right to fear them. An infant rat, upon smelling a strange adult male, will become immobile. However, a neonatal rat younger than 14 days (in other words, one still in the SHRP) will not become immobile: it hasn’t yet developed the machinery to feel, or possibly just to express, fear. If you remove the infant’s adrenals, so that it is unable to make corticosterone, then even when it matures to older than 14 days it will still not properly become immobile when exposed to the scary smell. Moreover, if you inject corticosterone into one of these pre-14 day rats, it will be able to develop the immobility behavior at age 14 days, just like a normal rat. [2] This suggests that corticosterone is responsible for the immobility behavior. However, if you remove the adrenals of a rat which has already developed the immobility behavior (one which is older than 14 days), it will continue to become immobile in the presence of the scary smell. [3] And if you inject extra corticosterone into a rat too young to have developed the immobility behavior, it will develop it early. [4] This suggests that corticosterone is responsible just for the development of the behavior, not for allowing it to actually happen at specific times once it has initially appeared.

What’s going on up in the brain while all this is happening? When infant rats are too young to express (or possibly feel) fear, are their amygdalas just failing to activate? When neurons in a particular brain region have been recently active, they contain a protein called c-fos. You can check a brain region for the prescence of extra c-fos to see if it has been doing anything in the recent past. This was done with young rats. Rats too young to have developed the fear response did not have amygdala activity (no extra amygdala c-fos) after exposure to the scary smell; if they were injected with corticosterone to cause them to develop the fear response early, then they did have amygdala activity; rats old enough to have developed the fear response did have amygdala activity; and rats whose adrenals were removed prior to developing the fear response did not have amygdala activity. [4] Unfortunately, this study does not appear to have looked at whether rats which were allowed to normally develop the fear response (intact adrenals), but then had their adrenals removed after initial development of the response, still showed amygdala activation. Perhaps that question has been answered elsewhere.

So what does all this mean for dogs? Do dogs have an SHRP? I found one unreferenced assertion that they do, but I have not yet found a study actually examining the canine SHRP. The SHRP does exist in various species, and it seems likely to me that it exists in the dog. Puppies start out fearless, and develop fear later. I suspect that a canine SHRP will prove to be an important part of socialization: the time that puppies don’t yet feel fear may be an important one for introducing them to lots of different kinds of people, so that they can learn that these people are a normal part of puppy life and are not to be feared later on.

The development of the HPA system has been studied in domesticated silver foxes — foxes selectively bred to not fear humans. (These foxes show surprising physical similarities to other domesticated animals in body shape and color, despite not having been bred for these features, leading to speculation that there is some general mechanism of domestication. That general mechanism of domestication is actually what I’d like to get at in a PhD project.) Researchers took two groups of foxes: domesticated foxes, and foxes bred for increased aggressiveness to humans. They tested them for behavioral reactions to humans and cortisol level increases after exposure to humans, at ages 30 days, 45 days, and 60 days. The aggressive foxes did not show aggressive behavior or cortisol spikes at 30 days, but they did show it at 45 and 60 days. The domesticated foxes, on the other hand, did not show aggressive behavior until 60 days, and their behavior at that time was described more as “defensive” than “aggressive.” They never showed the cortisol spike. [5]

Is this the same thing as a silver fox SHRP? I’m not sure that this study exactly gets at that, but it seems suggestive. Questions I’d like to ask about the SHRP in dogs are: Does the SHRP definitely exist in dogs? Is the SHRP length different in dogs and wolves? Does the length of the SHRP affect the socialization of the dog? Is the SHRP length different in different dog breeds? And, most important but most difficult to get at, does length of SHRP have anything to do with a dog’s fearfulness as an adult?

Wednesday, December 23, 2009

Today I got an email from a tech at my school, asking if the radiology department could use my dog Jack for a teaching exercise. My roommate had suggested Jack as a good dog for this particular class. He would be sedated, and students would radiograph (x-ray) him. In exchange for allowing them to practice on him, I would be able to request that any body part be radiographed, and have a free analysis of the image by a radiologist.

Jack does have marginally bad hips, but he hasn’t shown any discomfort lately, so I don’t think that getting rads of his hips is worth the annoyance to him of having to spend a day in the hospital and be sedated. Recovering from sedation isn’t awful, but it isn’t as fun as, say, not having been sedated in the first place, either. So the lure of free rads isn’t compelling to me.

I replied that I would be willing to let them use him, however, and this is why. I believe in use of student dogs, particularly mellow dogs like Jack who aren’t traumatized by being in the hospital for a day and being handled by strangers. What’s the alternative, after all? Students might learn these procedures on client dogs, who are actually sick or are less comfortable with strangers than Jack is. As my advisor once said, “When a veterinarian performs a procedure on your pet, how many times do you want him to have practiced that procedure on other animals?” The answer, of course, is “One thousand times.” We don’t want our animal to be the first.

Who else could vet students practice on besides their own dogs? One option would be to purchase animals for this use, or to use ex-research animals which are maintained on university campuses for this reason. Such animals are well cared for, of course, but it is not as good a life as being a pet. I believe that every dog should get to be loved by a human.

So Jack will volunteer yet again to help out. (He has already given blood to a study on hemangiosarcoma in golden retrievers, and gotten a cardiac ultrasound to help veterinary cardiologists better define values in normal dogs.) He will be annoyed. But if the visit goes like his previous volunteer appointments, he’ll get ice cream afterwards. That’s his payment for helping to advance veterinary medicine.

Monday, December 21, 2009

Recently I took my dog Jack along on a visit to a friend who was dog sitting. My friend’s resident dog has always gotten along with Jack pretty well, but this guest dog, Ally, had the habit of rushing at Jack when he entered the room, barking at him and generally behaving in a manner that alarmed all the humans involved. Ally was also hesitant around strange humans, in this case myself and my boyfriend. She would come up to us only gingerly, and was easy to scare away with sudden movements or loud noises.

My friend theorized that Ally’s issue with Jack was that she wanted to be the alpha dog, because dogs see the world in terms of a pack structure, like wolves. He felt the way to fix the problem was to let Ally know who was really boss, so whenever Ally rushed at Jack he would yell at her. The idea that a problem in dog training can be solved by asserting dominance is known as dominance theory; use of dominance theory has been publicized recently by Cesar Millan, the Dog Whisperer. A recent article in the Boston Globe addresses the controversy about Millan’s approach, and has this quote from Karen Pryor, who popularized clicker training, which is a very different approach:

...while his critics dispute Millan’s claim that domestic dogs are pack animals and should be treated as such, Pryor proposes that Millan’s hard-line message speaks to the real pack animal in the room. “We’re the ones who care very deeply about who’s boss and we don’t want to stop believing that humans are superior,’’ Pryor says. “We’re primates that have gone strongly in the direction of hierarchies. Dogs? They don’t care about that at all.’’

This quote really tickled me, even though in my opinion it goes a little too far. No offense to Pryor; I think her book Don’t Shoot the Dog should be required reading for any pet owner. But I think saying dogs don’t care about hierarchies at all may be overstating the case. In my house, it’s clear which dog is in charge, though the other two don’t seem to much care who comes second and who comes third. When I sat in on sessions with a veterinary behaviorist last year, I saw dogs diagnosed with dominance aggression, and I agreed with the diagnosis.

However, I do object to the blanket application of dominance theory to all problems in dog training. Wild dogs don’t run in packs the way wolves do; even if they did, to say that dominance issues are the answer to every canine behavioral problem is silly. Dogs are more complicated than that. In Ally’s case, I don’t think she was trying to show Jack that she was in charge; I think she was scared of him, and trying to deal with the situation in the best way she could come up with.

And who developed the idea that yelling is what makes a good leader? Are managers who regularly yell at their subordinates considered good bosses? My favorite bosses were always people who helped me solve my problems, not people who got angry at me when I couldn’t find a solution on my own.

The American Veterinary Society of Animal Behavior has a position statement on dominance theory, in which they provide a good framework for approaching its use in training. I do believe that overuse, or misuse, of dominance theory will gradually fade from the way we as a society manage our dogs, but for now, it seems to be hard for us as humans to let go of it.

Friday, December 4, 2009

Last night, before bed, I collected some saliva from my dog Jack and my roommate’s dog Casey. Jack has, by this point, become inured to the idea that occasionally I am going to grab his muzzle and stick a sponge on a stick inside his lip and swab around for two minutes. He was ready for bed when I did it last night and decided he might as well just doze through it.

Casey is another matter. When I cornered him and grabbed his muzzle, he freaked out.

Casey: OH MY GOD I THINK I AM GOING TO DIE

Me: Casey, I have done this to you five times. Why do you always act like it is going to hurt?

Casey: IT DOES HURT IT HURTS HORRIBLY THE PAIN IS BLINDING

Me: I have done it to myself. I know it doesn’t hurt.

Casey: IT HURTS DOGS! I CAN’T HOLD MYSELF UP ANY MORE

Me: I have done it to about 30 dogs and it didn’t seem to hurt any of them much.

Casey: Oh.

Most dogs are annoyed when I try to insert the swab, but then discover it doesn’t hurt and just deal with it. Casey inevitably squeezes his eyes shut, hyperventilates, and sometimes even collapses to the ground. (You might at this point ask “how do you know there isn’t something special about Casey?”, to which I would reply that, while there are certainly many special things about Casey, he also reacts this way to having his nails clipped, so I am pretty sure he is not experiencing any unusual physical sensations when I collect his saliva.)

I imagined Casey asking me “Why are you doing this to me?” and what my answer might be: “To find out how stressed you are.” As bizarre as that might be from a dog’s perspective, in fact my plan was to analyze his saliva to find out how much cortisol was in it. Cortisol has been called the “stress hormone,” and is an indicator of how much stress an animal is under.

Because I’ve never worked in a lab before, I wanted to do a few sample assays with unimportant data (i.e., saliva from my own dog that is easy to collect) before using the irreplaceable data collected from hospital dogs. It has taken me four months to successfully enroll 24 dogs; if I waste those samples, I am SOL. (Six dogs a month? Well, I’m a lot better at it than I used to be, and I had to take some time off in the middle. I am actually averaging 3-4 dogs a week at this point.) I performed my test assay this morning.

The cortisol assay is a kit costing a couple of hundred dollars which I purchased from the ever-helpful Salimetrics. It is an ELISA, or enzyme-linked immunoabsorbent, assay. The main equipment in the kit is a little plate (I was surprised by how tiny it was) made up of 96 wells, into each of which one might pour a very small amount of liquid. Each of the wells has some cortisol antibodies in the bottom (or so I’m told, since obviously antibodies are too small to see). These will grab on to any cortisol they see and hold on to it.

When I got into the lab this morning, I took the kit out of the fridge, and some older samples out of the freezer, so that they could warm up to room temperature. Then, while last night’s samples from Casey and Jack centrifuged, I sat down with the lab tech’s computer and figured out where everything was going to go on the plate. Aside from all my samples, I had to have room for the standards, which are samples given to you with the kit containing a known amount of cortisol. If your assay doesn’t tell you that the well with the 3.000 standard has about 3.000 µg/dL of cortisol in it, you know you’ve done something wrong. There are also the controls, which contain an arbitrary “high” and “low” amount of cortisol, and are also useful for telling you when you’ve messed up.

Then there are the “zero” and “blank” wells. The zero wells contain nothing but the diluent, or the liquid used to dilute some of the chemical agents in the kit. When the calculations are done at the end, you need to have this well to be able to tell the difference between a real effect and an effect caused by the diluent. And, finally, there are the blank wells, which don’t have any cortisol binding antibodies in them, and therefore will behave differently than the zero wells when the plate is read.

For me, the hardest part of the process is making sure that everything goes where it’s supposed to. This is more complicated than it sounds, involving lots of finding the right well in a small plate that is packed with small wells; remembering to leave space for a sample that I’m going to add later, after it has been diluted; getting the standards and controls in the right places; etc. Doing something that doesn’t require a lot of brainpower but does require a lot of precision, over and over and over, is hard for me.

When all the standards, controls, and samples were in place, and diluent was in the zero wells and the blank wells, I added conjugate to everything. The “conjugate” is cortisol which is attached to an enzyme. Of course, the whole point of the exercise is that you are putting in your saliva samples which contain cortisol, and now you’re adding this new source of cortisol as well. The two sources of cortisol are going to compete for the chance to bind to the cortisol-binding antibodies in the bottom of each well. When there is more cortisol in the sample, less of the conjugated cortisol (with its attached enzyme) can bind, and vice versa.

I put the plate with its samples and conjugate on to a rotator. This is a small device with a flat top which basically waves the plate around so that its contents mix. Every time I use it I am terrified that the plate is going to go flying off and spread my samples across the lab floor. It has not happened yet.

After 55 minutes for the whole competition thing to happen, I washed the plate off (four times), which includes blotting: picking the plate up, turning it upside down, and slamming it on to an absorbent pad. This is also a scary procedure, especially as sometimes strips of wells break off and you have to figure out in which direction to reattach them. Attach them upside-down, and you won’t know which sample is which.

At this point, in theory, the wells were mostly full of bound cortisol. Some of the cortisol, which came from the conjugate I added, had enzyme bound to it. Some of the cortisol, which came from my samples, did not. The wells which contained samples high in cortisol had less of the bound enzyme, and the wells which contained samples low in cortisol had more of the bound enzyme. Next I added TMB (tetramethylbenzidine), which reacts with the bound enzyme to change its color. Obviously, the wells with more bound enzyme will change to a different color than the wells with less.

More plate rotating is next, and then a brief time (sequestered in the dark) for the plate to let the reaction run its course. When I took the plate out of its light-proof bag at the end of this waiting period, the wells were all full of a lovely blue liquid, and different wells were visibly different shades. I added stop solution to stop the reaction, which immediately changed the color to yellow. And then I put the plate in the plate reader, which is a small machine attached to a Windows computer on the lab tech’s desk. It made some thumping noises as it determined the optical density of each well — its color, expressed as a number. It relayed these numbers to the waiting computer, which did the calculations to convert the numbers into concentrations, and I exported the results into Excel.

So how did I do? This was actually my second attempt at doing this assay. Both times, the results were fairly close to what I expected, based on the standards. However, part of what I wanted to figure out today was whether I could dilute small samples and still calculate values which were close to undiluted values. (In other words, if I dilute one sample by 50%, and multiple the calculated concentration by 2, will the result be similar to what I get when I put an undiluted version of that sample in a different well?) My dilutions were way off, so that’s something I need to figure out. I’m hoping to find a lab on campus which does these assays frequently, and ask if I can work with them for a little while, to get some experience.

By the way, the above implies that I competently performed this entire operation on my own, which is completely untrue: I worked under the close guidance of the extremely talented hospital lab technician. I am very lucky that she enjoys teaching; she told me today that she once considered being a school teacher. Her patience and good nature made the day much more enjoyable than it might have been.

Thursday, December 3, 2009

There have been some interesting developments recently in local meat processing in New England.

First, some background. I am a proponent of raising food animals on pasture on small farms. Specifics about why I believe in this approach are beyond the scope of this post; I was convinced by The Omnivore’s Dilemma by Michael Pollan and the Pew Report, also known as “Putting Meat on the Table: industrial farm animal production in America.” One of the main obstacles to success as a small scale food-animal farmer in New England, where I live, is the lack of processing operations (slaughterhouses) set up to handle small-scale farmers. There are plenty of people who want to raise animals on pasture and there are plenty of people who want to buy the meat or eggs, but there is a real bottleneck between the two, and building new slaughterhouses is expensive.

Mobile poultry processing units (MPPUs) are an inexpensive solution to the problem. MPPUs will actually come to your farm or to one near by to process your chickens. There are currently two MPPUs operating in Massachusetts, and more in surrounding New England states. Regulators are still somewhat wary of these units; they understand the need for them, but are more used to applying regulations to large scale plants than to slaughterhouses run out of flatbed trailers. Jen Hashley is the driving force behind one of the Massachusetts units. She has been working with regulators to make sure they’re happy with the unit, and looking for funding for getting more units built.

Recently, Grist reported that Whole Foods is considering getting into the mobile processing business by building their own fleet of MPPUs. Since Jen Hashley’s recent application for funding for new units was denied, this seems at first like a great solution to the problem. However, Whole Foods would allow use of the units only by farmers who have contracts with Whole Foods to grow chickens according to Whole Foods guidelines. Which is just contrary to the whole point of local food: that the farmer has the power to choose how he raises his animals, given his unique personal circumstances and beliefs. The consumer than has the power to choose which farmer to support, based on whether he agrees with that farmer’s choices. Not enough farmers around to allow the consumer to pick and choose? Well, that’s why it would be nice to have more mobile units, available to anyone who wants to pay to use them, to support the existence of more farmers making more varied choices.

I say:

Dear Whole Foods: Building MPPUs is a wise enterprise for you! However, you have already been criticized by Michael Pollan, the Foodie King, for your lack of support of local production, in his book The Omnivore's Dilemma and then in your opendiscussion with him. Allowing only limited access to your MPPUs will not be taken well by the locavore community, who will rightly see the move as an attempt to establish control overchicken production in this region. Why not allow open access to them? You’ll have fewer chickens grown exactly the way you want, of course, but aren’t consumers ready to pick and choose the kind of farmer they want to buy their chicken from? Some of these farmers will raise chickens your way, and you can sell those. Some of them won’t, and you don’t have to sell those — but why not give it a shot? Develop a labelling scheme which presents the relevant specifics to the consumer. On pasture? In a barn? Antibiotics? Organic feed? Different people care about different things. Some of us care a lot about choice.

Meanwhile, in Vermont, Walter Jeffries is responding to the lack of processing options by building an on-farm processing unit for his pastured pigs. He writes in great detail about the reasons he's choosing this solution. It’s a ballsy move. Building a slaughterhouse can cost literally millions of dollars. He has to try to balance doing so affordably, and complying with all the regulations necessary to convince the USDA to come inspect his meat. If the USDA refuses to inspect, he can’t sell his meat by the pound or across state lines. While MPPUs are the answer in some places, they aren’t the answer everywhere, and they are definitely not right for farmers who are raising animals of the four-legged variety.

I have no idea how either of these stories will end. Will Whole Foods’ choices, whatever they may be, support or limit local chicken production in New England? Will Jeffries be able to lure a USDA inspector to his plant when it’s done, or will it languish unused? It’s an interesting time to be watching news about local meat.

Sunday, November 22, 2009

It’s possible that twelve years of working as a programmer in the online publishing industry has sharpened my interest in open access, the idea that the public should have unlimited access to all scholarly publications. PLoS recently published "University Public-Access Mandates Are Good for Science," in which the author argues that, among other things, public access will help lift the “veil” from faculty research, creating greater public interest in (and funding for) research.

Open access is good for everyone who participates in research, really, except for the publishers. Current publication models based on fee for access, subscription or pay per view, aren’t compatible with open access. They might be compatible with a delayed access model, in which new content is embargoed for a year or so, then released to the public, and some people advocate those models. Others advocate the use of government funds to provide the services currently provided by the publishing industry.

What’s the publishing industry currently providing, and funding by charging for access to articles? Peer review is a service often mentioned, and it is extremely important. If a paper were published without having been reviewed by its author’s peers, no one would have any good way of judging its value. Loss of peer review might lead to chaos and the inability to find the wheat in the chaff. However, I am not convinced that this service can really be said to be provided by the publishing industry. The peers who review are, by definition, other researchers in the same community as the author of a submitted paper. Reviewers provide their services to a particular journal for free, as a resumé building move, to get a chance to see the very newest research before anyone else, and possibly out of the goodness of their hearts, to give back to the community. Would they be equally willing to donate their time to a well-known non-profit or university-affiliated group which organized review services, rather than a for-profit publishing group? I don’t see why not.

Indexing is also important. If a paper is no longer archived on a journal’s web site, but instead at a university’s institutional repository, would readers need to know the researcher’s university affiliation in order to find the paper? Readers could continue to use indexing services such as PubMed or Google Scholar, just as they do today. When presented with the title of an interesting article, my first step in finding the article’s text is never to try to find the journal’s site. I always visit Google Scholar first and let it show me my various options.

Journals are certainly useful for discovery purposes. I use Google Scholar to search for answers to specific questions, but I don’t use it to keep track of current research in a particular area. For that, I follow the latest articles published in particular journals, such as JAVMA or Applied Animal Behaviour Science. Certainly non-profit entities could serve similar purposes, aggregating links to important new recent articles. Authors might submit articles to these entities, just as they currently submit articles to journals.

The journal which publishes your article has a significant effect on how your article is perceived. If your article was accepted by the prestigious journal Nature, its importance will be perceived by readers to be greater than if the only journal you could find to accept it was some dinky little publication no one’s ever heard of. Again, non-profit entities could fill this purpose, but there would be some necessary lead time as some of them became established as high quality and others as, well, not so much.

What I haven’t seen mentioned as a useful contribution by the publishing industry is production editing — correction of spelling mistakes, bad grammar, and typos; modification of the paper’s layout to make it easier to read; conversion to PDF. I’m not sure if this is an important contribution in scholarly publishing or not. The articles I read seem to have more than their fair share of typos and grammatical mistakes compared to published books; it seems that in this area, content is valued much more highly than form. Perhaps this sort of work is something society would be willing to see lost.

This is all somewhat theoretical stuff at this point. Articles are being published open access (for example, in PLoS, the Public Library of Science), and indexing engines are indexing them. But the non-profit entities that I’m envisioning don’t seem to be springing up, and scholarly communities are still extremely dependent on publishing companies; the majority of articles are published in traditional for-pay journals. As a result, the goal of 100% release of scholarly articles under open access still feels somewhat distant. Some institutions are requiring their faculty to archive all publications in an open access institutional repositories; Harvard's new open access mandate is the most famous of these mandates. However, these institutional repositories have been described as “roach motels”: information goes in and doesn’t come out, due to poor searching facilities and lack of adherence to standards. (See “Institutional Repositories: thinking beyond the box.”) One previous comment on this blog mentioned arXiv, a highly successful open access repository in some of the hard sciences, but arXiv does not provide peer review services. A lot of the pieces of the puzzle exist, but they have not all been put together in a coherent way.

How can we organize the scholarly publishing community, which in fact consists of a huge number of communities in varied fields with their own publishing traditions? I’m glad it’s not my job to find the answer to that question. I suspect the answer will appear in its own time, and until then, I’ll keep following the various debates about open access, waiting to see what happens.

Saturday, November 21, 2009

Monday through Thursday evenings around five pm, I drive in to my school’s small animal hospital to collect spit from dogs. I’ve learned a lot about how to get spit out of a dog. It helps to have the right tools. If your subject is an adult human, you can ask them to drool into a cup. (Don’t actually spit! The manual doesn’t say why, but when I tried it, my spit contained long mucin strands which made it difficult to pipette up into a vial for storage.) If your subject doesn’t speak the same language as you do — mine don’t — or is otherwise recalcitrant, you might extract the spit with a small cotton rope. However, a new tool is now on the market: the Sorbette, a small sponge on a stick available from Salimetrics. (My endocrinology professor, upon hearing about Salimetrics, said: “An entire company dedicated to saliva? Who knew?”) Oh, wait. The Sorbette isn't actually all that new as technology goes: it turns out to just be an eye sponge. Not knowing much about eyes, I presume it is for adding things to or taking things away from them. But it's now also marketed to us spit collectors.

Salimetrics tells me to insert two Sorbettes under my subject's tongue and hold them there for a full minute. I should not swab. I did try this a few times. My subjects inevitably act like I have stuck a hot poker under their tongues, rolling their eyes and chewing madly. No Sorbettes have been damaged in this process, but it was only a matter of time. Also, the amount of saliva I actually acquired was too low for the assay I intended to perform on it.

So now I stick three Sorbettes in the side of the dog's mouth; this bothers them less, even with the greater mass. There also seems to be a fair amount of spit accumulating in the forward lower part of the dog’s mouth, between the outer edge of his teeth and inside his lip. And I do swab. The collection manual, when consulted, explained that swabbing is bad because it means you'll collect saliva from various different salivary glands, and so the levels of whatever you're testing might vary based on how much spit you get from which gland. This is a more important consideration for some other substances than for cortisol, which is what I care about.

Even so, the quantities of spit still occasionally leave something to be desired. My suspicion is that anxious dogs — which my study dogs are; that’s the point — just make less saliva than calm dogs, because their sympathetic nervous system is activated and telling them that making things which are useful for the process of digestion is not appropriate at this time. Eat later, worry about being stuck in the hospital now. In order to turn on the rest-and-digest parasympathetic nervous system, I have attempted waving treats under my subjects’ noses. Well, I’ve tried this once so far, and I did get a nice large sample that night. I left the treat behind with the dog; he was too anxious at the moment to ingest it, but I have hopes that he came around in my absence.

Once I have my sample, I balance it on a plate of ice and carry it upstairs to the clinical sciences laboratory, where I centrifuge it (3250 rpm, 15 minutes) so that it spins its way out of the sponge and into the tapered bottom of its vial. I then pipette it into a cryovial and secrete it in a -80° freezer. Done! When I have enough samples, I’ll perform an ELISA assay on them to find out how much cortisol is in each. I imagine I’ll report on that in detail here.

Saturday, November 14, 2009

After reading a post about how Strunk & White is a deeply weird book, I was inspired to pick it up and read it cover-to-cover, which I don't believe I'd ever actually done before. The book exhorts the reader, among many other wise statements, "Do not overstate":

When you overstate, the reader will be instantly on guard, and everything that has preceded your overstatement as well as everything that follows it will be suspect in his mind because he has lost confidence in your judgment or your poise.

I was briefly tempted to add this sentence to my email signature. It is the sentiment I would most like to convey to people who say things on the Internet. Too often, I read posts from people with whom I actually agree, but who make me flinch by overstating their case and poisoning the rest of their argument as a result. Certainly vet school has drilled into my head the rule that I should never make a statement that I can't back up, but even people who don't have graduate-level scientific training should be able to recognize that it's just bad debate style to make statements that overstep themselves. Less is more.

Thursday, November 12, 2009

Today, a newspaper local to me published a story about the devocalization debate in Massachusetts. The Massachusetts legislature is currently considering a bill which would make illegal the procedure for debarking dogs, or surgically altering their vocal cords to reduce the noise they make when barking. Various humane societies (including MSPCA-Angell) support the bill, because they feel that debarking is inhumane. The Massachusetts Veterinary Medical Association opposes the bill, because it takes away from veterinarians the ability to exercise their professional judgement.

I can see both sides of the issue. I don't know any details about the procedure itself, but I do know that when a dog barks excessively, there is usually some reason for it. Dealing with the symptom (barking) and not the underlying problem (separation anxiety, boredom, possibly something else) is not going to serve the owner or the dog well in the long run. The problem will manifest some other way. I don't know if this procedure is being used frequently and inappropriately in Massachusetts; I don't personally know any veterinarians who would perform it lightly, but I imagine that some such do exist in the state.

But should it be made illegal? I have to agree with the MVMA that the state should not be telling veterinarians how to do their jobs. Extenuating circumstances do occur, and I believe the decision to perform a procedure like this should be in the hands of the individual who spent four years earning his DVM (plus possible post-doctorate work in a residency program), and the owner of the dog, not in the hands of lawmakers who don't know the individual situation. Under the proposed bill, veterinarians will be able to apply for exemptions for "medical reasons," and only time would tell if behavior became recognized as a medical reason. But I don't like the idea of having to apply to the state to have a medical procedure done. I don't like the direction that takes us.

What do I propose as a solution? I think this problem should be solved by veterinarians. I think the MVMA should recognize that the problem is one that the citizens of this state currently find important, and should treat it seriously. A good first step could be forming a task force composed of representatives of the MVMA and of the groups who are in favor of this bill. This task force could provide a report on how often the debarking procedure is actually being performed, and under what circumstances. The task force could then identify problematic cases and discuss how they should have been handled differently, then discuss what options should have been available to that veterinarian and owner in that case. Are a significant number of veterinarians jumping to the surgery too quickly? Perhaps the MVMA can provide education to its members about the consequences of this procedure and some alternatives. Was the owner unaware of behavioral interventions? Could he not afford a trainer or a dog walker? Perhaps options should be made available for low-income people who cannot afford to deal with their animals' behavioral problems.

I would trust the MVMA and the MSCPA to find a solution to this problem, if they were able to work together. I'm not sure I trust the Massachusetts legislature to do so.

Tuesday, November 10, 2009

A researcher does a study. Based on the results, he comes to a conclusion about a small part of the nature of the universe. He writes up this conclusion and sends it to a journal. His peers review it and conclude that he did a good job in designing, implementing, and interpreting his study. In due time, his paper is published. The researcher receives non-monetary rewards.

Then, one day, he realizes he was wrong. He was wrong in a small way; he doesn't have to do another study to disprove his results, but he realizes he leapt to his conclusions. His interpretation was faulty. His data would have appeared different under very slightly changed study design. He considers letting his colleagues know. But how? He can't amend his paper. It is out there, as unrecallable as toothpaste from a tube. Does he write a letter to the journal? Post a note on his web page? Perhaps blog? None of these things will reach the audience that his original paper did.

It struck me, recently, how clumsy the communication protocol is that researchers use. They publish in papers. For a paper to change after publication, a slow, complicated machinery must be put in motion. A subsequent paper can show conflicting results. The paper can be found to be based on fraudulent data and recalled. Or the results can simply be gradually forgotten as science moves in a different direction. In any of these situations, change is slow.

I said as much in my bioethics class today. The professor asked, "How would you change things?" I suggested a world in which peer reviewed papers still existed, but another, non-peer-reviewed channel existed as well. We'd all know to take this channel's contents with a grain of salt, as they would be offered without the cautious filter of peer review. We would be much less likely to reference data from this channel in our papers. But we'd keep abreast of the conversations in it before completely trusting information we found in the more respectable peer-reviewed channel. It would be a place for back-and-forth, for something closer to a true conversation. (I've seen conversations in journals: two labs publishing an alternating series of articles that reference and refute each other, a dialog conducted over the course of years. It's rare and somewhat awestriking to observe.)

One of my classmates replied that I was idealistic. Well, sure. But this second channel is technologically very possible today. It might even currently be in the midst of developing out of the medium of science blogging. What would it take to start building mechanisms for allowing people to search it as easily as they currently use PubMed and Google Scholar to search the primary channel? I can't yet think where to start. Maybe all I need to do is sit back and wait, and watch the scientific information communication protocol evolve on its own, from its current clumsy elephantine form to something more nuanced and potentially graceful.

My roommate, RKt, has a 55 gallon marine fish tank. It is awesome. For more than a year, a small yellow fish has lived in it. I never saw this one, unlike the other fish; I'd forgotten he was even in there. He hides all the time. RKt, who interacts with the tank much more than I do, reports that she sees him about once every three months. He does not even come out at feeding time for delicious brine shrimp.

Last week, RKt brought home a new small yellow fish, of the same species, from a friend's tank which is being broken down. This new fish is very bold. He perches in high, easy to see places, on top of corals. He swims around fearlessly in open water. They have completely different personalities.

I remember our series of fire fish, little red and white gobies. I liked our first one so much that when he died I bought a second for the tank, and a third after that. (I have since given up, as they seem to have bad luck.) Firefish #2 (named S.S. Goby for his trait of rising up out of nowhere like a surfacing submarine) was best of friends with our burrowing shrimp. They shared what must have been a complex burrow structure under the live rock, living as congenial roommates. Firefish #3 (Mr. Goby) did not have much to do with the shrimp and liked to find other hiding places.

I'm intentionally anthropomorphizing, here, for amusement value. But it is clearly true to me that fish of the same species can behave in very different ways. That, to me, is personality.

Monday, November 9, 2009

Last week in journal club I presented "Prenatal exposure to maternal depression, neonatal methylation of human glucocorticoid receptor gene (NR3C1) and infant cortisol stress responses," by Oberlander et al., published in Epigenetics in 2008. This paper tries to get at one small part of the mechanism for how the in utero experience can affect a fetus, possibly even affecting the baby's personality.

Oberlander's study builds on earlier work done in rats. Researchers found that the offspring of particular rat dams were less fearful than average. Specifically, these rat moms were spending extra time licking and grooming their babies, and performing "arched-back nursing." They were dubbed "LG-ABN" dams (licking grooming, arched-back nursing.) Their babies acted less fearful in stressful situations, and had a blunted stress response on the HPA axis.

The HPA axis... This is in large part what I'm studying for my Masters project, so it's hard to limit myself to a short explanation of what it is. In short, one way in which one's brain (or part of it: the hypothalamus, the H in HPA) responds to stress is to send a message to the pituitary (P), which in turns sends a message to the adrenals (A). The adrenals release cortisol, which is known as the stress hormone. Cortisol is what researchers look for in your blood if they want to quantify how stressed you are. Stress out two animals, check their cortisol levels, conclude that the one with higher levels reacted more strongly to the stimulus — that's the formula for any number of experiments, including this one. (It is, as always with physiology, more complicated than that, but the idea that there's a correlation between increased cortisol levels and increased stress is a good start.)

So these babies of LG-ABN moms had a smaller cortisol spike in response to stress. Further work elucidated part of why that happened: the receptors to which cortisol binds in the brain send a message back to the top of the HPA to tell it to stop releasing cortisol ("that's enough, there's plenty out here!") in a negative feedback loop. These receptors (glucocorticoid receptors, abbreviated "GR") were in excessive supply in the brains of these less fearful rats, so the negative feedback loop worked well, and the rats' brains responded to rising cortisol levels by releasing less cortisol — with the result that the spike of cortisol was smaller in response to a stressful stimulus.

Meanwhile, the researchers also found that this trait of decreased fearfulness was not genetic in the normal sense. If they fostered baby rats from non-LG-ABN dams on LG-ABN dams, these babies who were not genetically related to the LG-ABN mom grew up to be less fearful, presumably simply by being raised by her. And they passed the trait on to their offspring! It turned out that the trait was being passed along epigenetically. We all learned in elementary school about genetic traits — getting brown eyes because you got brown eye genes from mom and dad. And we all know that genes are coded on DNA. Epigenetic changes involve not different genes, but changes to the higher-level structure of the DNA. Instead of involving changing the building blocks of the DNA (the genes), epigenetics involve changing the shape of the building, or sometimes tacking something new on to the outside of it.

In this case, it turned out that in order for GR to be produced (remember that receptor for cortisol, necessary for negative feedback?), the machinery for reading genes had to have free access to the GR gene itself. However, an area of that gene had become methylated — in other words, another object was sitting on it, blocking access. The machinery for reading genes couldn't read that gene as well, so fewer GRs were made. Fewer GRs meant less negative feedback and a more easily stressed baby rat.

That's all background. Oberlander, who wrote the paper I presented, wondered whether the same mechanism applied to humans. He knew that human mothers who are depressed during pregnancy often give birth to babies with more reactive HPA axes. Could that be because those babies had fewer GRs, as a result of methylation of the GR gene? He also wondered about the effects of SRI medication, such as Prozac, on this system.

82 pregnant women were enrolled in this study. 33 were taking SRI medication. All were tested using a scale for depression, which resulted in a numeric score; higher scores implied greater depression. Blood was drawn from the moms in their second and third trimesters, and when they gave birth. Blood was taken from the babies' umbilical cords at birth. Then the babies were tested at three months of age for their response to a mild stressor.

The researchers found that the babies of depressed moms did tend to have increased methylation of the GR gene, exactly in the spot that they expected. That increased methylation correlated with an increased cortisol spike when the babies were mildly stressed. SRI exposure in utero didn't have any effect on the size of the spike, although babies whose moms were medicated did tend to have lower cortisol levels in general.

This paper spoke to me on two levels. I enjoy reading about mechanisms; I like imagining how all these little machines in our bodies interact to form our personality and affect how we experience the world. I also liked the study's methods, because I'm interested in finding ways of learning about living individuals. I want to study dogs, so I want to find ways of looking into their brains figuratively, not literally. Examining changes in DNA extracted from a blood draw is cool — it's something I could potentially do to someone's pet, perhaps as part of a study aimed at understanding why some dogs are more easily stressed to the point of biting than are others.

I think that the people who attended journal club found the paper interesting. Two professors who were in attendance work in this area of genetics and behavior, and had useful input for me. One pointed out that the list of variables that the paper's authors checked for in the pregnant women was very small. (It consisted of things like age, whether this was a first pregnancy, whether the pregnancy ended in C-section, whether the woman smoked or drank.) She listed some other things she would have checked for, such as body weight (fat can apparently produce cortisol). She also noted that the baby's blood sample came from umbilical cord blood, which is actually a mix of infant and maternal blood. Also, different parenting strategies weren't taken into account — did depressed mothers treat their babies differently in some way? She concluded that we'd all like to be able to see useful DNA changes just by taking blood samples (which is precisely one of the things that drew me to this paper), but it's actually very hard to do so, so this paper's results should be taken very cautiously.

Saturday, November 7, 2009

I figure the first thing I say on this blog should be a basic orientation to who I am and where I'm coming from. I'm a veterinary and graduate student at Tufts Cummings School of Veterinary Medicine. I say both "veterinary and graduate" because I'm in a dual-degree program: I spent two years working on my DVM degree; this year I'm working on my MS degree in Comparative Biomedical Science; and in the fall of 2010, I'll return to the DVM program for its final two years.

My graduate work is in stress in hospitalized dogs: can we develop behavioral tools for helping us tell how stressed they are? And once we've done that, are there ways of making them less stressed?

What I really want to study, though, is dog brains — well, dog minds, since I like them operating more than I like them preserved. I feel very lucky to have managed to find a way to spend a year studying domestic dogs at all. When I came back to school after 12 years working in online publishing, veterinarians told me they only studied sick dogs, and researchers told me they only studied wild animals. So it's been hard to find a way to straddle that border — to study the normal, healthy behavior of a domesticated animal. (It sounds fairly wild in my house during Middle Aged Golden Retriever Wrestling Hour every night, with snarls, barks, and body slamming, but I guess that doesn't count as the opposite of domesticated.)

It's hard to say what this blog will become, but one of the things that seems to be lacking in veterinary medicine and scientific research is good communication with people operating in other walks of life. The new culture of science blogging is trying to overcome that division, so I'm throwing my cap into that fray.

About the Dog Zombie

Jessica Perry Hekman, DVM, PhD is fascinated by dog brains. She is a postdoctoral associate at the Broad Institute of MIT and Harvard, where she studies the genetics of dog behavior. Her interests include the stress response in mammals, canine behavior, canine domestication, shelter medicine, animal welfare, and open access publishing. You may learn more about Jessica at www.dogzombie.com, or email her at jph at dogzombie dot com. All opinions expressed here are her own.

For the animal shall not be measured by man… They are not brethren, they are not underlings: they are other nations, caught with ourselves in the net of life and time, fellow prisoners of the splendor and travail of the earth. (Henry Beston)